Ignite Creation Date:
2024-05-06 @ 11:49 AM
Last Modification Date:
2024-10-26 @ 12:50 PM
Study NCT ID:
NCT03603158
Status:
COMPLETED
Last Update Posted:
2018-07-30
First Post:
2018-07-18
Brief Title:
KCNH2 Polymorphisms on the QTc Interval in Kelantanese Malays Patients Receiving Methadone Maintenance Therapy MMT
Sponsor:
Universiti Sains Malaysia
Organization:
Universiti Sains Malaysia
Study Overview
Official Title:
Influence of KCNH2 Polymorphisms on the QTc Interval in Kelantanese Malays Patients Receiving Methadone Maintenance Therapy MMT in Malaysia
Status:
COMPLETED
Status Verified Date:
2018-07
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Methadone maintenance therapy MMT is one of the modalities to prevent HIV transmission among injected drug users particularly in opioid-dependent users However methadone-associated cardiotoxicity is one of the fatal adverse events that limit the widespread usage in certain groups of opioid-dependent patients This is a cross-sectional study aimed to investigate the association between 4 KCNH2 SNPs 1539CT in exon 6 of KCNH2 gene 1956TC in exon 8 of KCNH2 gene 2350CT in exon 9 of KCNH2 gene 2690AC Exon 11 of KCNH2 gene and prolongation of QTc interval in opioid-dependent Kelantanese Malays who are the recipients of Methadone Maintenance Therapy The investigators hypothesized that subjects with minor alleles of those 4 SNPs will have longer QTc intervals than those with major alleles adjusting for the effects of other confounding factors such as age and gender of the subjects plasma methadone trough levels hypokalemia hypocalcemia and hypomagnesemia The investigators also aimed to provide a model that will reliably predict the magnitude QTc based on the SNPs data and other covariates mentioned above This will greatly assist in identifying methadone recipients who are at risk of developing prolonged QTc or the more fatal torsade de pointes
Detailed Description:
Introduction
Methadone maintenance therapy MMT is one of the modalities to prevent HIV transmission among injected drug users particularly in opioid-dependent users However methadone-associated cardiotoxicity is one of the fatal adverse events that limit the widespread usage in certain groups of opioid-dependent patients
Study hypotheses aims
The investigators hypothesized that subjects with minor alleles of those 4 SNPs would have longer QTc intervals than those with major alleles adjusting for the effects of other confounding factors such as age and gender of the subjects plasma methadone trough levels hypokalemia hypocalcemia and hypomagnesemia The investigators also aimed to provide a model that will reliably predict the magnitude QTc based on the SNPs data and other covariates mentioned above This will greatly assist in identifying methadone recipients who are at risk of developing prolonged QTc or the more fatal torsade de pointes
Study Design and Sample Size Calculation
This is a cross-sectional study aimed to investigate the association between 4 KCNH2 SNPs 1539CT in exon 6 of KCNH2 gene 1956TC in exon 8 of KCNH2 gene 2350CT in exon 9 of KCNH2 gene 2690AC Exon 11 of KCNH2 gene and prolongation of QTc interval in opioid-dependent Kelantanese Malays who are the recipients of Methadone Maintenance Therapy The sample size was calculated using single-proportion formula and the information required was based on a similar prior study conducted among Singaporean Malay It was concluded that the sample size required is 105 patients Since eligible patients were lacking the convenience non-probability sampling method was used
During the initial visit relevant clinico-demographic details such as age gender history of drug addiction and psychiatric illnesses drug dependency patterns other drug usage and treatment-related issues for each patient were gathered Subsequently a validated Malay version of Subjective Opioid Withdrawal Scale SOWS questionnaire was administered to assess any opioid withdrawal symptoms experienced by study participants
Five 5 mls of blood was then withdrawn for each subject for the ascertainment of relevant biochemical profile serum potassium magnesium calcium plasma methadone trough levels and KCNH2 SNPs genotyping Drug screening for substances such as MDMA benzodiazepines methamphetamine cannabis marijuana were also carried out using urine dipstick test at urine collection point The colour and temperature of the urine were also recorded
QT measurement was obtained using calibrated Welch Allyn CP 50 ECG Electrocardiograph Welch Allyn Australia Pty Ltd New South Wales Australia machine printed at a paper speed of 25mms and voltage of 10mmmV QTc measurement was then manually calculated using Fredericias formula to correct for heart rate R-R interval All trained personnel who were responsible for obtaining QTc measurement from each patient were blinded to other information on serum biochemical profiles and methadone trough levels
KCNH2 Genotyping
The DNA was extracted according to procedures modified from Bethesda Research Laboratories based on the revisions made to the Brinboam and Doly method The quantity and quality of the extracted DNA were determined using NanoDrop ND-1000 Spectrophotometer NanoDrop Technologies Inc Wilmington USA with measurements done at 260 and 280 nm The integrity of the extracted DNA was determined using 2 agarose gel electrophoresis performed at 70 Volts for 90 minutes
The DNA in all samples were amplified for all 4KCNH2 SNPs were performed using 2-step nested allele-specific multiplex polymerase chain reaction PCR The primers both forward and reverse were designed according to the published sequence for KCNH2 NC_00000713 To improve primer specificity mismatch at its 3 ends that were specific to either the variant sequence or wild-type DNA sequence at the specified locus was made to the primers Besides the primers were also designed and manipulated to differentiate between the different single nucleotide changesalleles during PCR amplification To verify primers specificity the BLAST program at NCBI httpwwwncbinlmnihgov blast was used
In the first multiplex PCR exon 689 and 11 were amplified under the following condition pre-denaturation at 95C for 1 minute followed by 25-cycle of denaturation at 95C for 15 seconds annealing for 65C for 15 seconds and extension at 72C for 10 seconds Upon full 25-cycle completion final extension phase lasted for 72C for 7 minutes The PCR products amplicons were then resolved using 2 agarose gel electrophoresis at 130 Volts for 90 minutes
The PCR products of the first multiplex PCR were then used as templates for second PCR which targeted the 4 respective SNPs region for amplification This was performed under the following conditionpre-denaturation at 95C for 1 minute followed by 25-cycle of denaturation at 95C for 15 seconds annealing for 69C for 30 seconds and extension at 72C for 4 seconds Upon full 25-cycle completion final extension phase lasted for 72C for 7 minutes The PCR products amplicons were then resolved using 2 agarose gel electrophoresis at 130 Volts for 90 minutes
The first products of the amplified regions in the Exons 6 8 9 and Exon11 were subsequently submitted for direct DNA sequencing QIAquick PCR purification kit Qiagen USA was used for purification of the PCR products DNA sequencing was carried out by applying 3130XL genetic analyzer DNA sequencer ABI USA The results were compared with the published sequences for KCNH2 in the NCBI accession number NC_00000713
Statistical Analysis
The select equally likely or more extreme samples SELOME version of the Fishers exact test was employed to examine whether the distribution of the SNP alleles and genotype follow the Hardy-Weinberg Equilibrium HWE assumption Those SNPs that significantly deviated from the HWE assumption were dropped from further analysis
To examine the associations between the 4SNPs and QTc interval measured as continuous variable and build a statistical model that can reliably predict QTc intervals simple and multiple linear regression methods were used Age and gender of the patients plasma methadone trough levels serum potassium calcium and magnesium were treated as confounding factors whose effects on QTc were adjusted for
Methadone maintenance therapy MMT is one of the modalities to prevent HIV transmission among injected drug users particularly in opioid-dependent users However methadone-associated cardiotoxicity is one of the fatal adverse events that limit the widespread usage in certain groups of opioid-dependent patients
Study hypotheses aims
The investigators hypothesized that subjects with minor alleles of those 4 SNPs would have longer QTc intervals than those with major alleles adjusting for the effects of other confounding factors such as age and gender of the subjects plasma methadone trough levels hypokalemia hypocalcemia and hypomagnesemia The investigators also aimed to provide a model that will reliably predict the magnitude QTc based on the SNPs data and other covariates mentioned above This will greatly assist in identifying methadone recipients who are at risk of developing prolonged QTc or the more fatal torsade de pointes
Study Design and Sample Size Calculation
This is a cross-sectional study aimed to investigate the association between 4 KCNH2 SNPs 1539CT in exon 6 of KCNH2 gene 1956TC in exon 8 of KCNH2 gene 2350CT in exon 9 of KCNH2 gene 2690AC Exon 11 of KCNH2 gene and prolongation of QTc interval in opioid-dependent Kelantanese Malays who are the recipients of Methadone Maintenance Therapy The sample size was calculated using single-proportion formula and the information required was based on a similar prior study conducted among Singaporean Malay It was concluded that the sample size required is 105 patients Since eligible patients were lacking the convenience non-probability sampling method was used
During the initial visit relevant clinico-demographic details such as age gender history of drug addiction and psychiatric illnesses drug dependency patterns other drug usage and treatment-related issues for each patient were gathered Subsequently a validated Malay version of Subjective Opioid Withdrawal Scale SOWS questionnaire was administered to assess any opioid withdrawal symptoms experienced by study participants
Five 5 mls of blood was then withdrawn for each subject for the ascertainment of relevant biochemical profile serum potassium magnesium calcium plasma methadone trough levels and KCNH2 SNPs genotyping Drug screening for substances such as MDMA benzodiazepines methamphetamine cannabis marijuana were also carried out using urine dipstick test at urine collection point The colour and temperature of the urine were also recorded
QT measurement was obtained using calibrated Welch Allyn CP 50 ECG Electrocardiograph Welch Allyn Australia Pty Ltd New South Wales Australia machine printed at a paper speed of 25mms and voltage of 10mmmV QTc measurement was then manually calculated using Fredericias formula to correct for heart rate R-R interval All trained personnel who were responsible for obtaining QTc measurement from each patient were blinded to other information on serum biochemical profiles and methadone trough levels
KCNH2 Genotyping
The DNA was extracted according to procedures modified from Bethesda Research Laboratories based on the revisions made to the Brinboam and Doly method The quantity and quality of the extracted DNA were determined using NanoDrop ND-1000 Spectrophotometer NanoDrop Technologies Inc Wilmington USA with measurements done at 260 and 280 nm The integrity of the extracted DNA was determined using 2 agarose gel electrophoresis performed at 70 Volts for 90 minutes
The DNA in all samples were amplified for all 4KCNH2 SNPs were performed using 2-step nested allele-specific multiplex polymerase chain reaction PCR The primers both forward and reverse were designed according to the published sequence for KCNH2 NC_00000713 To improve primer specificity mismatch at its 3 ends that were specific to either the variant sequence or wild-type DNA sequence at the specified locus was made to the primers Besides the primers were also designed and manipulated to differentiate between the different single nucleotide changesalleles during PCR amplification To verify primers specificity the BLAST program at NCBI httpwwwncbinlmnihgov blast was used
In the first multiplex PCR exon 689 and 11 were amplified under the following condition pre-denaturation at 95C for 1 minute followed by 25-cycle of denaturation at 95C for 15 seconds annealing for 65C for 15 seconds and extension at 72C for 10 seconds Upon full 25-cycle completion final extension phase lasted for 72C for 7 minutes The PCR products amplicons were then resolved using 2 agarose gel electrophoresis at 130 Volts for 90 minutes
The PCR products of the first multiplex PCR were then used as templates for second PCR which targeted the 4 respective SNPs region for amplification This was performed under the following conditionpre-denaturation at 95C for 1 minute followed by 25-cycle of denaturation at 95C for 15 seconds annealing for 69C for 30 seconds and extension at 72C for 4 seconds Upon full 25-cycle completion final extension phase lasted for 72C for 7 minutes The PCR products amplicons were then resolved using 2 agarose gel electrophoresis at 130 Volts for 90 minutes
The first products of the amplified regions in the Exons 6 8 9 and Exon11 were subsequently submitted for direct DNA sequencing QIAquick PCR purification kit Qiagen USA was used for purification of the PCR products DNA sequencing was carried out by applying 3130XL genetic analyzer DNA sequencer ABI USA The results were compared with the published sequences for KCNH2 in the NCBI accession number NC_00000713
Statistical Analysis
The select equally likely or more extreme samples SELOME version of the Fishers exact test was employed to examine whether the distribution of the SNP alleles and genotype follow the Hardy-Weinberg Equilibrium HWE assumption Those SNPs that significantly deviated from the HWE assumption were dropped from further analysis
To examine the associations between the 4SNPs and QTc interval measured as continuous variable and build a statistical model that can reliably predict QTc intervals simple and multiple linear regression methods were used Age and gender of the patients plasma methadone trough levels serum potassium calcium and magnesium were treated as confounding factors whose effects on QTc were adjusted for
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None